The concentration, oxygenation, and flow characteristics of blood cells in the microvasculature profoundly affect nourishment and thermal regulation of surrounding tissues. If these blood variables become abnormal, then tissue health may be threatened. Measurement of these basic blood parameters can reveal information about severe burns, reconstructive surgeries, peripheral vascular disease (PVD), brain metabolism and functional heterogeneity, internal bleeding, tumor vasculature, lymph flow, and ischemia. The goal of this competing continuation grant is to improve, validate and apply diffuse optical spectroscopy for the investigation of blood dynamics in deep tissues. New instrumentation has been built that combines for the first time photon correlation technology for studies of tissue internal motions such as blood flow, with diffuse photon density wave spectroscopic tools for studies of tissue absorption and scattering. The new instrument makes possible noninvasive separation of blood flow, blood oxygenation scattering, and blood volume tissue responses. While the experimental results to date are useful and reveal promise for eventual research and clinical application, the applicants have identified several target areas wherein continued progress is critical to transfer this methodology from the developmental stage toward clinical applications. The proposed research has three interrelated components. A theoretical component develops better models specific to physiological problems the applicants plan to investigate in murine tumors, rat brain, and on human limbs. A second component validates the instruments and analysis in well defined tissue phantoms and physiological models for which the biological response is either well understood and/or is measured with another experimental technique. The last component investigates how transferability of the composite optical diagnostics can be used in: (1) State-of-the-art animal studies of tumor necrosis factor and cerebral metabolic rate, and (2) Ongoing clinical studies of peripheral vascular response.